A non‐chromatographic method for the removal of endotoxins from bacteriophages

Abstract

The Ff filamentous bacteriophages show potential as a new class of therapeutics, displaying utility in materials science as well as pharmaceutical applications. These phages are produced by the infection of E. coli, a Gram-negative bacterium which unavoidably sheds endotoxins into the extracellular space during growth. Since endotoxin molecules are highly immunoreactive, separation from the phage product is of critical importance, particularly those developed for human therapeutic use. The properties of M13, one of the Ff group, present a purification challenge chiefly because the standard scalable method for endotoxin removal from proteins-anion exchange chromatography-is not applicable due to pI similarity between the particles. This article examines the potential of polyethylene glycol (PEG)-NaCl precipitation as a scalable method for the separation of endotoxins from phage M13. Precipitation of M13 by 2% (w/v) PEG 6 000, 500 mM NaCl reduced endotoxin contamination of the phage product by 88%, but additional precipitation rounds did not maintain this proportional decrease. Dynamic light scattering was subsequently used to determine the effectiveness of a detergent to disassociate endotoxin molecules from M13. As a result, PEG-NaCl precipitation was supplemented with up to 2% (v/v) Triton X-100 to improve separation. A 5.7 log10 reduction in endotoxin concentration was achieved over three rounds of precipitation whilst retaining over 97% of the phage. This method compares favorably with the well-known ATPS (Triton X-114) technique for endotoxin removal from protein solutions.

Peer Review #2 of "Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks (v0.1)"

“…Unfortunately, neither the PoT protocol nor the traditional methods were effective at significantly reducing bacterial endotoxins from phage preparations, and additional purification steps were required. Numerous methods and commercially available kits are available for the removal of bacterial endotoxins (Merril et al, 1996;Boratyński et al, 2004;Merabishvili et al, 2009;Oślizło et al, 2011;Branston, Wright & Keshavarz-Moore, 2015), yet many of these are either highly specific, time consuming, laborious, or expensive. Szermer-Olearnik & Boratyński recently proposed the use of an organic solvent to successfully reduce bacterial endotoxin from phage lysates (<20 EU•ml -1 ).…”

“…However, the Phage On Tap protocol was not able to effectively reduce bacterial endotoxins, likely due to our ultrafiltration-based approach and the large aggregate size of endotoxins. Numerous endotoxin removal procedures and commercial kits are available (Boratyński et al, 2004;Merabishvili et al, 2009;Oślizło et al, 2011;Batista et al, 2013;Branston, Wright & Keshavarz-Moore, 2015), yet many of these methodologies lack generality, are time consuming, or are cost-prohibitive. Recently, the successful reduction of endotoxins (<20 EU•ml -1 ) from phage lysates was reported by extraction with organic solvents (Szermer-Olearnik & Boratyński, 2015).…”

Response: Commentary: Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro

“…Despite the suggestion to the contrary provided by the Dufour et al commentary, the reduction and quantification of endotoxin in phage preparations remains an active area of research (Branston et al, 2015; Szermer-Olearnik and Boratyński, 2015) as more effective, less labor intensive, and perhaps most importantly for commercial development, more cost effective methods are sought. However, this highlights the disparity between preparative methods for phage assessment and once again shows the urgent need for standardized methods and acceptance criteria, similar to those employed in antibiotic and biocide development to be developed.…”

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“…Unfortunately, neither the PoT protocol nor the traditional methods were effective at significantly reducing bacterial endotoxins from phage preparations, and additional purification steps were required. Numerous methods and commercially available kits are available for the removal of bacterial endotoxins (Merril et al, 1996;Boratyński et al, 2004;Merabishvili et al, 2009;Oślizło et al, 2011;Branston, Wright & Keshavarz-Moore, 2015), yet many of these are either highly specific, time consuming, laborious, or expensive. Szermer-Olearnik & Boratyński recently proposed the use of an organic solvent to successfully reduce bacterial endotoxin from phage lysates (<20 EU•ml -1 ).…”

“…However, the Phage On Tap protocol was not able to effectively reduce bacterial endotoxins, likely due to our ultrafiltration-based approach and the large aggregate size of endotoxins. Numerous endotoxin removal procedures and commercial kits are available (Boratyński et al, 2004;Merabishvili et al, 2009;Oślizło et al, 2011;Batista et al, 2013;Branston, Wright & Keshavarz-Moore, 2015), yet many of these methodologies lack generality, are time consuming, or are cost-prohibitive. Recently, the successful reduction of endotoxins (<20 EU•ml -1 ) from phage lysates was reported by extraction with organic solvents (Szermer-Olearnik & Boratyński, 2015).…”